Poly(amino acids), including peptides and proteins, are typically detected and characterized using gel electrophoresis, by solution quantitation assays or by detection on solid supports, such as filter membranes. Small amounts of protein or other poly(amino acids) are generally not visible to the naked eye, and must be stained before they can be localized and identified.
Two of the most common methods of staining poly(amino acids) in gels are COOMASSIE Brilliant Blue (CBB) staining and silver staining. For particular poly(amino acids), silver staining is approximately 100- to 1000-fold more sensitive than CBB staining, but both methods have disadvantages. In contrast, the use of luminescent reagents, such as fluorescent, phosphorescent or chemiluminescent reagents, for protein detection offers the possibility of greatly enhanced sensitivity and increased linear quantitation range, while simultaneously increasing the ease of use of the staining reagent.
The use of exemplary fluorescent stains, such a styryl and merocyanine dyes, for poly(amino acids) in gels (e.g. SYPRO® Red and SYPRO® Orange dyes of Invitrogen Corporation), on membranes or other supports, and in solution (U.S. Pat. No. 5,616,502 to Haugland et al., hereby incorporated by reference) is very rapid, relatively insensitive to poly(amino acid) composition, does not require destaining, and is more than an order of magnitude more sensitive than CBB staining.
Other fluorescent stains (e.g. SYPRO® Ruby [Invitrogen Corporation], Flamingo™ Fluorescent Gel Stain [Bio-Rad Laboratories], Deep Purple™ Total Protein Stain [GE Healthcare], Krypton™ Protein Stain [Pierce Biotechnology]) offer even better sensitivity, but require long multi-step staining procedures. These commercial fluorescent protein stains, with the exception of SYPRO Ruby, are excited relatively weakly by ultraviolet light. They are optimally imaged using visible light laser-based scanners as opposed to relatively simple and inexpensive instrumentation utilizing ultraviolet transillumination.